Toll样受体9在肾衰后血管钙化中的作用及机制

The incidence of vascular calcification in dialysis patients is 2 to 5 times of healthy persons, and vascular calcification is the most important risk factor for the morbidity and mortality of cardiovascular disease in patients with chronic renal failure (CRF). The disorders of calcium and phosphorus metabolism, increased FGF-23 and vitamin D levels, secondary hyperparathyroidism play important roles in vascular calcification of patients with renal failure. Recent studies show that chronic inflammation plays an important role in vascular calcification. Our previous data showed that the inflammatory response was increased in CRF rats. The present preliminary data showed that bacteria translocation was present in CRF animals, and inhibition of intestinal bacteria and clearance of intestinal LPS significantly reduced vascular calcification in the CRF animals. More importantly, the serum from CRF rats aggravated Pi-induced vascular smooth muscle cells(VSMCs) calcification after correction of calcium ion and phosphorus levels, and this effect was blocked by Toll-like receptor 9 (TLR9) inhibitor. Therefore, we got the propose that intestinal bacteria translocation-caused chronic inflammation promoted vascular calcification in individuals with chronic renal failure through activation of TLR9. The project was planned to study the role of bacterial CpG-DNA in high phosphate-induced VSMC calcification with or without co-culture with macrophages. Therefore, to explore the underlying mechanisms of CpG-DNA/TLR9 signal in promoting vascular calcification including the direct effects of them on VSMCs and the indirect effects through activating macrophages and producing cytokines (TNFalpha and IL-1beta et al). And finally using knockout mouse model of renal failure to confirm the important role of bacteria translocation and TLR9 in chronic inflammation and vascular calcification of individuals with CRF, and then help to provide new ideas and strategies for the prevention and treatment of vascular calcification in CRF patients.

透析病人血管钙化发生率是正常人的2-5倍，是其血管性疾病发病和死亡的重要危险因素。近年研究表明慢性炎症是加重血管钙化的重要因素。申请者前期工作证明肾衰动物存在肠道细菌易位、全身炎症水平增加；抑制肠道细菌可显著减轻肾衰后血管钙化；校正钙磷水平后，肾衰大鼠血清仍可以加重离体培养的血管平滑肌钙化,并且这一作用可被TLR9特异性阻断剂所阻断。因此提出设想：TLR9介导了肠道细菌易位加重肾衰后血管钙化的作用。拟在高磷诱导的血管平滑肌细胞（VSMC）钙化模型中，探讨细菌组分LPS和CpG-ODN作用于其受体TLR4和TLR9对VSMC的促钙化作用，以及其可能的分子机制;并利用TLR4、TLR9基因敲除小鼠肾衰模型，在体阐明TLR9在肾衰后血管钙化发病中的重要作用及其机制。不仅从整体和分子水平上进一步阐明肾衰后血管钙化的发病机制,证实TLR9信号在其中的重要作用,为防治肾衰后血管钙提供新理论和靶点。

血管钙化（vascular calcification）是指钙磷复合物在血管壁的异位沉积。是患动脉粥样硬化、高血压、糖尿病血管病变、血管损伤和慢性肾病等疾病的病人中普遍存在的共同的临床病理表现。透析病人血管钙化发生率是正常人的2-5倍，是其血管性疾病发病和死亡的重要危险因素。近年研究表明慢性炎症是加重血管钙化的重要因素。申请者前期工作证明肾衰动物存在肠道细菌易位、全身炎症水平增加；抑制肠道细菌可显著减轻肾衰后血管钙化；校正钙磷水平后，肾衰大鼠血清仍可以加重离体培养的血管平滑肌钙化,并且这一作用可被TLR9特异性阻断剂所阻断。因此提出设想：TLR9介导了肠道细菌易位加重肾衰后血管钙化的作用。拟在高磷诱导的血管平滑肌细胞（VSMC）钙化模型中，探讨细菌组分LPS和CpG-ODN作用于其受体TLR4和TLR9对VSMC的促钙化作用，以及其可能的分子机制;并利用TLR4、TLR9基因敲除小鼠肾衰模型，在体阐明TLR9在肾衰后血管钙化发病中的重要作用及其机制。不仅从整体和分子水平上进一步阐明肾衰后血管钙化的发病机制,证实TLR9信号在其中的重要作用,为防治肾衰后血管钙提供新理论和靶点。在本项目的资助下，本课题组阐明了（1）肾衰大鼠存在肠道细菌易位，肠系膜淋巴结细菌培养菌落数量明显增加，血中可检测到细菌16SrDNA增加，给予抗生素治疗清除肠道细菌后，肾衰引起的血管钙化明显减轻肾衰大鼠；（2）Toll-Like receptor 9激活可以促进高磷诱导的人血管平滑肌细胞钙化和成骨分化；（3）Toll-Like receptor 4激活可以促进高磷诱导的人血管平滑肌细胞钙化和成骨分化；（4）抗生素治疗可以通过抑制TLR9信号减轻肾衰大鼠血管钙化；（5）肾衰大鼠血清促进离体培养的VSMC钙化主要是通过 激活TLR9，而非TLR4，说明在肾衰大树中是opG-DNA而非LPS促进血管钙化；（6）证明BMP-2的上调介导了TLR9激活促进血管平滑肌细胞钙化的作用。从而得出结论：肠道细菌易位释放细菌CpG-ODN，通过激活单核巨噬细胞和/或血管平滑肌细胞上的TLR9，引起肾衰患者全身和血管局部慢性炎症反应，进而加重肾衰患者血管钙化。本项目研究成果和结论为进一步阐明肾衰后血管钙化的发生发展提供了新的理论和认识，为临床防治血管钙化提供新的思路和靶点。